Synthetic polynucleotide coding for human lactoferrin, vectors, cell and transgenic plants containing it

Abstract

A synthetic polynucleotide encoding human lactoferrin, modified with respect to the natural gene so as to maximize its expression in vegetals, on the basis of the preferential use of the codons is described. Moreover, the vectors containing such sequence, that having regulation elements activated in a controlled way determine its tissue- and stage-specific expression are further described. The vegetal cells and the plants transformed with the afore mentioned vectors, as well as the production processes of functional foods, vegetal milks, and human lactoferrin, utilizing them are also described.

1. A plant expression cassette, wherein said expression cassette expresses non-degraded human lactoferrin in seed, said cassette comprising a gene encoding human lactoferrin comprising the sequence SEQ ID NO: 23, said gene being operatively linked to the sequence SEQ ID NO: 21 and a leader sequence encoding a signal peptide of the sequence SEQ ID NO: 26. 
     
     
       2. The plant expression cassette according to  claim 1 , wherein said leader sequence has the sequence SEQ ID NO: 13. 
     
     
       3. The plant expression cassette according to  claim 1 , wherein said gene encoding human lactoferrin has the sequence SEQ ID NO: 1. 
     
     
       4. A recombinant DNA vector comprising the plant expression cassette of  claim 1 . 
     
     
       5. The recombinant DNA vector according to  claim 4 , wherein said leader sequence has the sequence SEQ ID NO: 13. 
     
     
       6. The recombinant DNA vector according to  claim 4 , wherein said gene coding for human lactoferrin has the sequence SEQ ID NO: 1. 
     
     
       7. A method for using the vector according to  claim 4  for the transformation of vegetal cells comprising:
 transferring said vector in competent  agrobacterium  cells; and 
 transforming said vegetal cells with the  agrobacterium  cells obtained from said transferring. 
 
     
     
       8. A vegetal cell including the vector of  claim 4 . 
     
     
       9. A cellular aggregation obtained from cells according to  claim 8 . 
     
     
       10. The cellular aggregation according to  claim 9  wherein said aggregation is a callus, and wherein said callus is capable of regenerating a transgenic plant. 
     
     
       11. A transgenic plant, comprising the expression cassette of  claim 1 , said plant expressing in-seed the non-degraded protein human lactoferrin. 
     
     
       12. The transgenic plant according to  claim 11 , said plant being selected from the group consisting of solanaceae, cereals and leguminosae. 
     
     
       13. The transgenic plant according to  claim 12 , said plant being selected from the group consisting of soya, tobacco and rice. 
     
     
       14. A method for using the vector according to  claim 4  for transformation of vegetal cells comprising:
 subjecting said cells to bombing with a biolistic system; and 
 biolistically transforming said cells with said vector. 
 
     
     
       15. A method for production of human lactoferrin extracts comprising:
 collecting seeds of the transgenic plant according to  claim 11 ; and 
 grinding said seeds in contact with an extraction buffer to produce said human lactoferrin extract. 
 
     
     
       16. A method of using the transgenic plant according to  claim 11  for production of non-degraded human lactoferrin comprising:
 collecting seeds of said transgenic plant; 
 grinding said seeds in contact with an extraction buffer; and 
 purifying said non-degraded human lactoferrin therefrom. 
 
     
     
       17. A method for production of human lactoferrin-containing flour comprising:
 collecting seeds of the transgenic plant according to  claim 11 ; and 
 grinding said seeds into human lactoferrin-containing flour. 
 
     
     
       18. A method for production of vegetal milk containing human lactoferrin comprising:
 collecting seeds of the transgenic plant according to  claim 11 ; and 
 grinding said seeds into flour and suspending same to produce said vegetal milk. 
 
     
     
       19. A method for using the vector according to  claim 5  for the transformation of vegetal cells comprising:
 transferring said vector in competent  agrobacterium  cells; and 
 transforming said vegetal cells with the  agrobacterium  cells obtained from said transferring. 
 
     
     
       20. A vegetal cell including the vector of  claim 5 . 
     
     
       21. A cellular aggregation obtained from cells according to  claim 20 . 
     
     
       22. The cellular aggregation according to  claim 21  wherein said aggregation is a callus, and wherein said callus is capable of regenerating a transgenic plant. 
     
     
       23. A transgenic plant, comprising the expression cassette of  claim 2 , said plant expressing in-seed the non-degraded protein human lactoferrin. 
     
     
       24. The transgenic plant according to  claim 23 , said plant being selected from the group consisting of solanaceae, cereals and leguminosae. 
     
     
       25. The transgenic plant according to  claim 24 , said plant being selected from the group consisting of soya, tobacco and rice. 
     
     
       26. A method for using the vector according to  claim 5  for transformation of vegetal cells comprising:
 subjecting said cells to bombing with a biolistic system; and 
 biolistically transforming said cells with said vector. 
 
     
     
       27. A method for production of human lactoferrin extracts comprising:
 collecting seeds of the transgenic plant according to  claim 23 ; and 
 grinding said seeds in contact with an extraction buffer to produce said human lactoferrin extract. 
 
     
     
       28. A method of using the transgenic plant according to  claim 23  for production of non-degraded human lactoferrin comprising:
 collecting seeds of said transgenic plant; 
 grinding said seeds in contact with an extraction buffer; and 
 purifying said non-degraded human lactoferrin therefrom. 
 
     
     
       29. A method for production of human lactoferrin-containing flour comprising:
 collecting the seeds of the transgenic plant according to  claim 23 ; and 
 grinding said seeds into human lactoferrin-containing flour. 
 
     
     
       30. A method for production of vegetal milk containing human lactoferrin comprising:
 collecting seeds of the transgenic plant according to  claim 23 ; and 
 grinding said seeds and suspending same to produce said vegetal milk. 
 
     
     
       31. A method for using the vector according to  claim 6  for the transformation of vegetal cells comprising:
 transferring said vector in competent  agrobacterium  cells; and 
 transforming said vegetal cells with the  agrobacterium  cells obtained from said transferring. 
 
     
     
       32. A vegetal cell including the vector of  claim 6 . 
     
     
       33. A cellular aggregation obtained from cells according to  claim 32 . 
     
     
       34. The cellular aggregation according to  claim 33  wherein said aggregation is a callus, and wherein said callus is capable of regenerating a transgenic plant. 
     
     
       35. A transgenic plant, comprising the expression cassette of  claim 3 , said plant expressing in-seed the non-degraded protein human lactoferrin. 
     
     
       36. The transgenic plant according to  claim 35 , said plant being selected from the group consisting of solanaceae, cereals and leguminosae. 
     
     
       37. The transgenic plant according to  claim 36 , said plant being selected from the group consisting of soya, tobacco and rice. 
     
     
       38. A method for using the vector according to  claim 6  for transformation of vegetal cells comprising:
 subjecting said cells to bombing with a biolistic system; and 
 biolistically transforming said cells with said vector. 
 
     
     
       39. A method for production of human lactoferrin extracts comprising:
 collecting seeds of the transgenic plant according to  claim 35 ; and 
 grinding said seeds in contact with an extraction buffer to produce said human lactoferrin extract. 
 
     
     
       40. A method of using the transgenic plant according to  claim 35  for production of non-degraded human lactoferrin comprising:
 collecting seeds of said transgenic plant; 
 grinding said seeds in contact with an extraction buffer; and 
 purifying said non-degraded human lactoferrin therefrom. 
 
     
     
       41. A method for production of human lactoferrin-containing flour comprising:
 collecting the seeds of the transgenic plant according to  claim 35 ; and 
 grinding said seeds into human lactoferrin-containing flour. 
 
     
     
       42. A method for production of vegetal milk containing human lactoferrin comprising:
 collecting seeds of the transgenic plant according to  claim 35 ; and 
 grinding said seeds and suspending same to produce said vegetal milk.